Electron-counting MicroED data with the K2 and K3 direct electron detectors.
Electron-counting MicroED data with the K2 and K3 direct electron detectors.
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DOI：
10.1016/j.jsb.2022.107886
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发表时间：
2022-12
影响因子：
3
通讯作者：
Gonen, Tamir
中科院分区：
文献类型：
--
作者：
Clabbers, Max T. B.;Martynowycz, Michael W.;Hattne, Johan;Nannenga, Brent L.;Gonen, Tamir
微晶电子衍射 (MicroED) 使用电子冷冻显微镜 (cryo-EM) 在样品连续旋转过程中收集小晶体的衍射数据。由于硬件和方法开发的进步，数据质量在过去十年中不断提高，甚至可以从头开始确定大分子结构。理想情况下，适合电子衍射的探测器应具有快速读出功能，以在电影模式下记录数据，并在低曝光率下具有高灵敏度，以准确报告强度。直接电子探测器由于其灵敏度和速度而常用于冷冻电镜成像，但尽管它们可用，但通常不用于衍射。衍射实验的主要问题是动态范围和重合损耗，如果通量超过检测器的计数率，这将破坏测量结果。在这里，我们描述了使用 K2 和 K3 直接电子探测器在电子计数模式下的仪器设置和低曝光 MicroED 数据收集，并表明积分强度可以有效地用于解析 1.2 Å 和 2.8 Å 分辨率之间的两种大分子的结构。尽管 K3 研究中没有使用光束挡板，但我们没有观察到相机损坏。由于这些相机已在许多冷冻电镜设施中使用，这为无法使用 MicroED 专用设施的用户提供了机会。
Microcrystal electron diffraction (MicroED) uses electron cryo-microscopy (cryo-EM) to collect diffraction data from small crystals during continuous rotation of the sample. As a result of advances in hardware as well as methods development, the data quality has continuously improved over the past decade, to the point where even macromolecular structures can be determined ab initio. Detectors suitable for electron diffraction should ideally have fast readout to record data in movie mode, and high sensitivity at low exposure rates to accurately report the intensities. Direct electron detectors are commonly used in cryo-EM imaging for their sensitivity and speed, but despite their availability are generally not used in diffraction. Primary concerns with diffraction experiments are the dynamic range and coincidence loss, which will corrupt the measurement if the flux exceeds the count rate of the detector. Here, we describe instrument setup and low-exposure MicroED data collection in electron-counting mode using K2 and K3 direct electron detectors and show that the integrated intensities can be effectively used to solve structures of two macromolecules between 1.2 Å and 2.8 Å resolution. Even though a beam stop was not used with the K3 studies we did not observe damage to the camera. As these cameras are already available in many cryo-EM facilities, this provides opportunities for users who do not have access to dedicated facilities for MicroED.
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DOI：
10.1107/s0907444910045749
发表时间：
2011-04
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Winn MD;Ballard CC;Cowtan KD;Dodson EJ;Emsley P;Evans PR;Keegan RM;Krissinel EB;Leslie AG;McCoy A;McNicholas SJ;Murshudov GN;Pannu NS;Potterton EA;Powell HR;Read RJ;Vagin A;Wilson KS
通讯作者：
Wilson KS
影响因子：
5
作者：
Takaba K;Maki-Yonekura S;Inoue S;Hasegawa T;Yonekura K
通讯作者：
Yonekura K
影响因子：
48
作者：
Nannenga, Brent L.;Shi, Dan;Leslie, Andrew G. W.;Gonen, Tamir
通讯作者：
Gonen, Tamir
影响因子：
64.8
作者：
Nakane T;Kotecha A;Sente A;McMullan G;Masiulis S;Brown PMGE;Grigoras IT;Malinauskaite L;Malinauskas T;Miehling J;Uchański T;Yu L;Karia D;Pechnikova EV;de Jong E;Keizer J;Bischoff M;McCormack J;Tiemeijer P;Hardwick SW;Chirgadze DY;Murshudov G;Aricescu AR;Scheres SHW
通讯作者：
Scheres SHW
电子计数和束诱导运动校正可实现近原子分辨率的单粒子冷冻电镜。
影响因子：
48
作者：
Li, Xueming;Mooney, Paul;Zheng, Shawn;Booth, Christopher R.;Braunfeld, Michael B.;Gubbens, Sander;Agard, David A.;Cheng, Yifan
通讯作者：
Cheng, Yifan